Submitted to: Journal of Integrative Plant Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/28/2005
Publication Date: 11/1/2005
Citation: Li, J., Qi, R., Hu, L., Mattoo, A.K., Li, N. 2005. Cleavage of the carboxyl-terminus of leacs2, a tomato 1-aminocyclopropane-1-carboxylic acid synthase isomer, by a 64-kda tomato metalloprotease produces a truncated but active enzyme. Journal of Integrative Plant Biology. 47:1352-1363. Interpretive Summary: Plant hormones regulate plant growth, development and senescence. One such hormone is a gaseous hydrocarbon, ethylene. Among myriad processes that ethylene regulates, its role in plant aging, fruit ripening and cell death is pivotal. A key protein that controls the production of ethylene in plants is called ACC synthase. Until this protein is activated plants cannot make copious amounts of ethylene. Therefore, studies on this protein at the gene and protein levels have been intense in recent years. Previously, we had shown that this protein is cleaved at its carboxy terminus and that the carboxy terminus region is critical for its dimeric structure and its activity. The nature of the activity that cleaves ACC synthase is not known. In this report, we identify and purify a candidate protein that uses metal ions for its catalysis and cleaves ACC synthase. We further demonstrate that the truncated form of ACC synthase does not lose enzymatic activity. This work opens up new strategies to look at the function of the cleavage of ACC synthase in regulating plant development, fruit ripening and plant cell death. It should be of interest to horticulturists, biochemists, biotechnologists and industry interested in how proteins are degraded and what is their fate.
Technical Abstract: 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS (LeACS2) into 52-, 50- and 49-kDa truncated isoforms in ripening tomato (Lycopersicon esculentum Mill. cv. Cooperation 903) fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys438, Glu447, Lys448, Asn456, Ser460, Ser462, Lys463, and Leu474, but does not cleave the N-terminus of LeACS2. Four C-terminus-deleted (26–50 amino acids) LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located onthe N-terminal side of the phosphorylation site Ser460. The results indisputably confirmed the presence of cleavage sites within the region between the '-helix domain (H14) and Ser460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.